| Objective:Coronary tortuosity is defined as continuous or spiral curvature of epicardial vessels,which is a common vascular abnormality in angiography,but its etiology and clinical significance are not yet clear.From the biomechanical point of view,vascular tortuosity can lead to the disturbance of local hemodynamic environment characterized by flow separation or recirculation and low oscillatory wall shear stress that have been extensively demonstrated to associate with the initiation and progression of atherosclerosis.However,its effects on vascular calcification,the process of ectopic deposition of mineral matrix in the vascular wall,are still unclear.In order to further understand the influencing factors of coronary tortuosity and its impact on vascular calcificationis defined as abnormal deposits of mineral matrix in the vessel wall,we explored them from two levels of clinical and basic experiments,so as to improve our understanding of the disease,and learn the deep meaning behind its existence.The specific research contents are as follows:(1)The clinical risk factors affecting coronary tortuosity;(2)The effects of shear stress on intimal calcification.Specific researches are as follows:PartⅠ:The risk factors affecting coronary tortuosityMethods:The case data of patients who underwent coronary CTA examination at Zhongda Hospital Affiliated to Southeast University from January 1,2020 to June 9,2022 were retrospectively collected,including general information,coronary CTA examination results,cardiac color Doppler ultrasound results,and biochemical indicators.The CAC score was calculated by special software and quantified as Agatston score.After screening by inclusion and exclusion criteria,1533 patients were included in the study.Coronary tortuosity is defined as the presence of at least three consecutive curvatures of more than 45°measured during systole or diastole of a major epicardial coronary artery.The population prevalence of coronary tortuosity in patients with coronary CTA was calculated.Tortuous prevalence of coronary branches and their concomitant branches in patients of different genders and ages was analyzed and compared.Logistic regression analysis was used to explore the risk factors of coronary branch tortuosity.Results:In this study,the prevalence of coronary artery tortuosity was 38.6%,among which,the prevalence rate of single-vessel tortuosity was 22.5%,the prevalence of combined two-vessel tortuosity was 12.6%,and the prevalence of three-vessel tortuosity was 3.6%.Among single-vessel and multi-vessel lesions,the highest prevalence of LAD tortuosity was 13.4%,and the lowest prevalence of LCX&RCA tortuosity was 0.7%.The prevalence of coronary tortuosity in males was lower than in females(30.9%and 46.5%,respectively).Among them,LAD was the most commonly involved artery,with a prevalence of 11.3%in males and 15.6%in females.The prevalence of LCX&RCA was the lowest,0.5%and 0.8%,respectively.The prevalence of tortuosity in each branch of the coronary arteries in both genders(male vs.female)was LAD 23.8%vs.34.0%,LCX 13.1%vs.25.7%,RCA 10.4%vs.10.1%.Patients without coronary tortuosity were in the 18-29 age group,while patients with single-vessel and multi-vessel lesions were mostly in those aged 70-80 years.In different age groups,the LAD was most involved,followed by the LCX,and the RCA was the least affected.For men aged 60-69,the prevalence of coronary tortuosity is the highest(11.1%)among single-vessel and multi-vessel lesions,while for women aged70-80,that prevalence is the highest(46.5%).Univariate logistic regression analysis found that age,BMI,hypertension,diabetes and Agatston score were the main factors affecting coronary tortuosity,but after adjusting confounding factors,it was found that the age,BMI,hypertension and Agatston score were the independent risk factors influencing coronary tortuosity.Elderly patients(LAD:OR 1.04,95%CI 1.03-1.05,P<0.001;LCX:OR 1.03,95%CI 1.02-1.04,P<0.001;RCA:OR 1.03,95%CI1.01-1.04,P<0.001)and patients with hypertensive(LAD:OR 1.61,95%CI 1.23-2.09,P<0.001;LCX:OR 1.46,95%CI 1.08-1.97,P=0.014;RCA:OR 2.47,95%CI 1.62-3.75,P<0.001)and moderate Agatston score(LAD:OR 1.49,95%CI1.01-2.18,P=0.043;LCX:OR 2.32,95%CI 1.07-5.09,P=0.034;RCA:OR 0.91,95%CI 0.81-1.01,P=0.041)were particularly prone to coronary tortuosity.Obese patients(LAD:OR 0.55,95%CI 0.37-0.82,P=0.003;LCX:OR 0.51,95%CI 0.30-0.85,P=0.010;RCA:OR 0.46,95%CI 0.23-0.85,P=0.021)were less likely to have coronary tortuosity than those with normal weight.Female(OR 1.36,95%CI 1.06-1.74,P=0.014)and EF value(OR 1.01,95%CI 1.00-1.03,P=0.038)were risk factors for LAD tortuosity;Female(OR 2.09,95%CI 1.58-2.78,P<0.001)and diabetes(OR1.59,95%CI 1.12-2.19,P=0.004)were risk factors for LCX tortuosity;Smoking(OR1.79,95%CI 1.09-2.95,P=0.025)was significantly correlated with RCA tortuosity.Conclusion:The prevalence of coronary tortuosity is higher,and more prevalent in males than females.Coronary tortuosity tends to increase with age.Age,BMI,hypertension and Agatston score were the common influencing factors affecting coronary tortuosity,while gender,smoking,EF value and diabetes were the independent risk factors in different coronary branch tortuosity.PartⅡ:Effect of shear stress on intimal calcificationMethods:Laminar flow and oscillatory shear flowgenerated by Ibidi flow system(Ibidi,Germany)were applied to HUVECs with shear stress of 12 dyn/cm~2(LSS),0.5±6 dyn/cm~2(OSS),respectively.After 24 h,RNA-seq was used to detect genes with differential expression,and GO enrichment analysis was performed to screen target gene.Subsequently,the effect of this gene on HUVEC calcification after being silenced or overexpressed was confirmed by Alizarin red staining,Western blotting,RT-q PCR,immunofluorescence,and co-immunoprecipitation.Results:Under shear loading,RNA‐seq analysis identified 3583 differentially expressed genes,among which 1956 genes were up‐regulated and 1627 genes were down‐regulated in the OSS group compared to the LSS group.GO enrichment analysis showed that the differentially expressed genes were mainly enriched in shear stress and ossification related pathways.Additionally,Mst1 was screened as the target gene.Under the condition of LSS intervention,the m RNA expression level of Mst1 increased with the extension of time.In contrast,the m RNA expression level of Mst1 was significantly decreased with the extension of OSS intervention time.Consistent with the RT-q PCR results,the protein expression level of Mst1 also decreased over time after OSS intervention.In addition,compared with the LSS group,the immunofluorescence intensity of Mst1 was decreased in the OSS group.Next,HUVECs were transfected with lentivirus-mediated short hairpin RNA(sh RNA)targeting Mst1.Compared with NC-sh RNA group,the protein expression levels of RUNX2,BMP2 and Msx2 in the Mst1-sh RNA group were increased.When MST1was overexpressed,compared with the NC-OE+β-GP group,the protein levels of RUNX2,BMP2,and Msx2 were decreased.Consistent with this,the number of calcified nodules,and the calcium content of HUVEC in the Mst1-OE+β-GP group were significantly decreased.In addition,co-localization of Mst1 and RUNX2 was observed by immunofluorescence staining.Inhibition of RUNX2 synthesis and degradation by CHX and MG132,respectively,further confirmed that overexpression of Mst1 reduced the protein stability and enhanced the ubiquitination and degradation of RUNX2.Knockdown of WWP2 was achieved by lentiviral vector-mediated sh RNA expression,the protein expression level of RUNX2 was not significantly changed under Mst1 overexpression.CO-IP results showed that overexpression of Mst1promoted the binding of WWP2 to RUNX2,while knockdown of Mst1 inhibited the binding of WWP2 to RUNX2,thereby affecting the occurrence of VC.Conclusion:The decrease of Mst1 induced by OSS is closely related to the occurrence of VC.Overexpression of Mst1 can promote the ubiquitination and degradation of RUNX2 through WWP2,thereby reducing calcium deposition in EC. |
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